Morphological, ontogenetic, and molecular investigations of freshwater hypotrich ciliates from China revealed a new genus Heterodeviata gen. nov. (Protista: Ciliophora), and a novel limnetic population of Deviata multilineae Author Song, Wenya Institute of Evolution & Marine Biodiversity, Ocean University of China, ºingdao 266003, China & Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China Author Dong, Jingyi Institute of Evolution & Marine Biodiversity, Ocean University of China, ºingdao 266003, China Author Lu, Xiaoteng Department of Biology, Shenzhen MSU-BIT University, Shenzhen 518172, China Author Al-Farraj, Saleh A. Zoology Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia Author Song, Weibo Institute of Evolution & Marine Biodiversity, Ocean University of China, ºingdao 266003, China & Laoshan Laboratory, ºingdao 266237, China Author Hines, Hunter N. Harbor Branch Oceanographic Institute, Florida Atlantic University, Fort Pierce, Florida, 34946, USA Author Luo, Xiaotian Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China luoxiaotian@ihb.ac.cn text Zoological Journal of the Linnean Society 2023 2023-06-30 199 1 263 279 http://dx.doi.org/10.1093/zoolinnean/zlad044 journal article 10.1093/zoolinnean/zlad044 0024-4082 8326400 Deviata Eigner, 1995 Deviata multilineae Zhang et al., 2022 ( Fig.7A–N ) Deposition of voucher specimens : Three voucher slides (No. SWY2020051501/1–3) with protargol-stained specimens were deposited in the Laboratory of Protozoology, Ocean University of China , ºingdao, China . Cell size 150–175 × 40–60 μm (160 × 50 μm on average) in vivo ( N = 6), with ratio of length to width in vivo ranging from 2.8 to 3.9; about 165 × 83 μm (140–185 × 55–116 μm), length:width ratio about 2.0:1 (1.6–2.9:1) asser protargol impregnation. Cell flexible but not contractile, outline elongate elliptical, usually with anterior part wider than the posterior one ( Fig. 7A–D, F–H ). Algae in food vacuole rendered cell yellow/ greenish at low magnification ( Fig. 7A, F–H ). Cytoplasm colourless, with numerous colourless globules (0.75–1.5 μm) and many transparent colourless inclusions (2–11 μm), most being spherical and located in the middle of the cell ( Fig. 7E, I ). Cortical granules absent. Four macronuclear nodules, ellipsoidal-shaped, 15–18 × 13–16 μm in vivo , usually two micronuclei, each one usually located between a pair of macronuclear nodules ( Fig. 7I, K, N ). Contractile vacuole about 15 μm when full, located at the equatorial level, near less cell margin ( Fig. 7A, B–D, H ). Collecting canals absent. Locomotion by slowly floating in water, sometimes crawling around the substratum. No cell division, cysts, or conjugation were observed. The adoral zone of membranelles occupies 28% of cell length in protargol preparations, composed of 23–35 membranelles ( Table 1 ). Paroral and endoral consisted of single-rowed dikinetids, nearly parallel to each other in most cells (intersected in two out of 15 cells examined), with the former shorter than the laưer ( Fig. 7J, M ). The cytopharyngeal fibres extend posteriorly from the cytostome ( Fig. 7J ). Length of cirri in vivo about 11–13 μm. Three frontal cirri, usually one buccal cirrus located right to the anterior end of the paroral and anterior of the endoral (two buccal cirri observed in three out of 20 cells), three to seven parabuccal cirri behind the right frontal cirrus ( Fig. 7J, M, N ). Three frontoventral cirral rows, frontoventral cirral row 1 (FVR1) commences at right of right frontal cirrus and terminates at posterior 15% of cell length. In six out of 15 cells, FVR1 consisting of two parts: the anterior part terminates behind the proximal end of AZM, the posterior part commences at the right of several last cirri of anterior part ( Fig. 7J, N ). Another two frontoventral cirral rows terminate at the rear end of the cell ( Fig. 7J, N ). Eight to eleven less marginal rows, most of them located on the dorsal side; two or three right marginal rows, most of them distributed on the ventral side ( Fig. 7J, K, L, N ). Figure 4. Early to middle morphogenetic stages of Heterodeviata sinica asser protargol impregnation. A, macronuclear nodules of a very early divider (ventral view), showing replication band of macronuclear nodules (arrows). B, oral primordium of the opisthe (arrow). C, developed oral primordia (arrow). D–F, ventrolateral (D, E) and dorsolateral (F) view of the same early divider, showing incomplete adoral zone of membranelles (arrow in D, E), the intrakinetally formed marginal anlagen and dorsal kinety anlage 1. G, H, ventral (G) and dorsal (H) view of the same early-middle divider, showing the dorsal kinety 2 anlage formed to the right of the right marginal anlage 2. I, J, ventral (I) and dorsal (J) view of the same middle divider, denoting anlagen I–IV differentiating into cirri, arrowhead in (I) shows the parabuccal cirrus. DḎ1, 2, dorsal kineties anlagen 1, 2; FC, frontal cirri; FVR, frontoventral cirral row; LMA1, 2, less marginal anlagen 1, 2; Ma, macronuclear nodules; Mi, micronuclei; RMA1, 2, right marginal anlagen 1, 2; UMA, undulating membrane anlage. Scale bars: 20 μm (A–C), 50 μm (D–J). Three long dorsal kineties (DK), dorsal bristles inconspicuous in vivo . DK1 with 9–17 dikinetids, DK2 with 17–27 dikinetids, DK3 with 11–17 dikinetids. Caudal cirri absent ( Fig. 7K ). 18S rRNA gene sequence and phylogenetic analyses ( Figs 8 , 9 ) The two new 18S rRNA gene sequences obtained in this study were deposited in GenBank. Their lengths, GC-content, and accession numbers are as follows: Heterodeviata sinica 1571 bp, 45.59%, OP537913; Deviata multilineae 1630 bp, 45.77%, OP537914. Figure 5. Morphogenetic process of Heterodeviata sinica asser protargol impregnation. A, showing the oral primordium of the opisthe. B, ventral view of a very early divider, showing oral primordium of the opisthe (arrowhead). C–E, ventro-lateral (C, D) and dorsolateral (E) views of the same early divider, in this stage, anlagen I–IV (C), two right marginal anlagen (E) formed, adoral zone of membranelles incompletely formed (arrowhead in C), and arrowhead in (D) shows the parental endoral beginning to disintegrate. F, G, ventral (F) and dorsal (G) views of two middle dividers, arrowheads in (G) showing dorsal kinety anlage 2. H, I, ventral (H) and dorsal view (I) of two middle dividers, in this stage, anlagen I–IV dedifferentiating into cirri (H), and two bristles of dorsal kinety 2 formed (arrowheads in H, I). J, dorsal view of a late divider, showing one macronuclear nodule dedifferentiated into two ones. K, L, ventral views of two late dividers, arrowheads in (K) and (L) indicate the buccal and parabuccal cirrus, separately. I–IV, anlagen I–IV; DK1, dorsal kinety 1; DḎ1, dorsal kinety anlage 1; FVR, frontoventral cirral row; LMA1, 2, less marginal anlagen 1, 2; LMR1, 2, less marginal rows 1, 2; Ma, macronuclear nodules; Mi, micronuclei; OP, oral primordium; RMA1, 2, right marginal anlagen 1, 2; RMR1, 2, right marginal rows 1, 2; UM, undulating membranes. Scale bars: 50 μm (F, L), 20 μm (G, I). Phylogenetic trees based on 18S rRNA gene sequence data using BIandMLanalysesaremostlycongruent, therefore,onlytheMLtree isshownwithsupportsfrombothalgorithms ( Fig.2A ). Accordingto the 18S rRNA gene tree, all deviatids group together in a large clade with high to full support (97%ML/1.00 BI), in which Heterodeviata sinica is placed in a subclade including D. parabacilliformis , D. rositae , and Pseudosincirra longicirrata with low support (69%ML/0.54BI). However, the position of H. sinica is not robust due to the low support for the cluster of H. sinica , D. parabacilliformis , and D. rositae in the ML tree (50%) and the incongruent topology in the BI tree ( Fig. 2C ). Furthermore, the systematic relationship of Heterodeviata with related genera is shown in Figure 2B . Comparing the two new 18S rRNA sequences with seven available deviatid sequences ( Fig. 8 ), Heterodeviata sinica differs from the other deviatid sequences by 31–38 nucleotide positions (corresponding to 97.5–97.9% similarity). It is noteworthy that for the sequence of Perisincirra sp. (KY855575, submiưed by Nitla from Pisa University), the morphological characterization was unavailable, but there were no nucleotides differences from Deviata brasiliensis , which suggests that Perisincirra sp. (KY855575) was misidentified, and probably is conspecific with Deviata brasiliensis . More detailed information about the nine deviatid sequences are presented within Figure 8 .